A classical approach to understanding the mechanisms of electrical signaling in neurons has been the use of temperature- sensitive (TS) paralytic mutants of Drosophila. These mutants develop normally at permissive temperature and can be shifted to restrictive temperatures to examine the physiological function of a specific gene product. Drosophila is ideal for this purpose given that it is exothermic and amenable to sophisticated genetic, molecular, biochemical, electrophysiological, and behavioral analysis. Our previous work has extended some early studies on TS paralytic mutants by examining the role of the comatose (comt) gene product in synaptic transmission. Our analysis has shown that the comt gene product, dNSF1, functions in the priming of docked vesicles for fast, calcium-triggered exocytosis. To broaden our investigation of synaptic mechanisms, we have conducted genetic screens for new mutations affecting synaptic transmission, including a screen for genetic modifiers of comt. One enhancer of comt is a TS paralytic allele of a calcium channel alpha1 subunit gene, cacophony (cac). Synaptic physiology in this mutant, termed cacTS2, shows that the cac gene product represents the primary calcium channel alpha1 subunit responsible for transmitter release at neuromuscular synapses. The proposed experiments will advantage of a number of new mutants recovered in a genetic screen for modifiers of cacTS2. The rapid TS paralytic phenotype of cacTS2, as well as the central role of voltage-gated calcium channels in neurotransmitter release, have provided another central starting point from which to expand our genetic analysis of synaptic transmission. Our screen for genetic modifiers of cacTS2 has been highly successful, resulting in the recovery of both new cac alleles and extragenic mutations in other genes functioning in synaptic transmission. The proposed experiments will capitalize on the success of this screen to pursue further genetic analysis of the functions and interactions of cac-encoded calcium channels in synaptic transmission.